The Epidemiology Branch is conducting a number of birth defect studies in collaboration with the Health Research Board and Trinity College, Dublin, Ireland. The main objective of these studies is to determine the relationship between folate and birth defects. The birth defects studied to date are neural tube defects (NTDs), oral clefts, congenital heart defects, Down syndrome and omphalocele. These studies focus on biochemical factors in the area of folate metabolism, and on genetic mutations in folate related genes associated with birth defects. Recent work has expanded to include the biochemical pathways related to birth defects. On going work is examining pathways in healthy young students of Irish genetic background (the Trinity Student Study) with several goals. These include identifying genetic factors using genome wide association study data that influence these pathways and defining how environmental influences such as smoking, alcohol and the use of supplements change the biochemical milieu. Neural tube defects (NTDs) are common birth defects (1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. We have been testing genetic variants for association with NTD for a long time including examining the biological importance of genetic variants. Dr. Anne Parle-McDermott led our group in a recent investigation of the biological importance of a dihydrofolate reductase (DHFR) variant. This investigation was important because DHFR is a key enzyme in the absorption of folic acid. This investigation showed that an insertion/deletion in the DHFR gene does not interfere with folic acid absorption. It has been suggested that low maternal choline status is a risk factor for NTD, although the data are conflicting. This is a critical question to address because of the possibility that adding choline supplements to women's diets prior to conception could decrease NTD rates in their offspring. We measured total choline and betaine concentrations in Irish women carrying affected fetuses and women carrying unaffected control fetuses. There was no significant difference in choline or betaine concentrations suggesting that taking choline supplements would not reduce NTD rates. We have measured tryptophan (Trp) pathway chemical concentrations in the over 2500 student volunteers in the Trinity Student Study. Trp is affected by numerous chronic diseases; however, little information is available regarding normal concentrations of Trp metabolites or how life style factors influence them. We demonstrated that gender is an important factor in interpreting Trp pathway metabolites. Moreover, use of oral contraceptives, alcohol consumption and use of vitamin B6 containing supplements also had a significant effect on Trp metabolites. A similar investigation looked at genetic and environmental factors that affect vitamin B6 (pyridoxal phosphate and related vitamers). We found that genetic variants in tissue nonspecific alkaline phosphatase (ALPL) affect serum concentrations of vitamin B6 and raise the possibility that they may affect the functional status of this vitamin. This study has been published in the Journal of Nutrition. Dr. Lawrence Brody at NHGRI is leading an investigation using our NTD cases to look for familial risk factors. This study is ongoing. We also published (Pangilinan et al.) a report following up on folate genes that were plausible candidates for association with NTDs. It was noteworthy that none of the 24 candidate polymorphisms reached statistical significance after correction for multiple testing. This study illustrates the problem of searching for genetic associations. Even with a large NTD population like ours, false negatives may occur. Conversely, it is possible that none of the candidates originally found in a previous study were true positives. We have had a productive collaboration with investigators at the University of Michigan to identify genetic factors that modify clotting. We previously identified new genetic factors that affect von Willebrand factor. We have finished a second collaboration examining plasminogen that has been published in Blood. Plasminogen is a key factor in the fibrinolytic system. We showed that both smoking and female gender affect plasminogen and that there is a previously unrecognized genetic component that influences plasminogen concentrations. Both Dr. Ruzong Fan and Dr. Aiyi Liu, research statisticians on our project, have used our work and data to identify important statistical research questions. Their major area is looking for more efficient and powerful methods to identify true associations in genome-wide investigations. This work has produced several important papers noted in the bibliography. These will be discussed by Dr. Fan and Dr. Liu in their reports.